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Methods for Promoting Extracellular Expression of Proteins in Bacillus subtilis Using a Cutinase

a technology of bacillus subtilis and extracellular expression, applied in the field of gene engineering, enzyme engineering and microbial engineering, can solve the problems of cumbersome and costly extraction process, limited application in the food industry, and low efficiency of the subsequent extraction process, so as to promote extracellular secretion of recombinant proteins, simplify the downstream purification process, and save costs

Active Publication Date: 2022-05-05
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The invention provides a method for promoting the extracellular secretion of recombinant intracellular enzymes in a B. subtilis system. By co-expressing a cutinase mutant, the method successfully achieves the extracellular expression of several enzymes, such as xylose isomerase, 4,6-α-glucosyltransferase, 4-α-glucosyltransferase, trehalose synthase, and branching enzyme. This method simplifies the downstream purification process and saves costs, making it great for large-scale production. Overall, the invention provides a way to efficiently produce industrial proteins.

Problems solved by technology

The subsequent extraction process is cumbersome and costly, so it has become the goal of researchers to find an effective way to simplify the downstream extraction steps and reduce the purification cost of products.
However, E. coli is not considered a food safety grade strain, so its application in food industry is limited.
When the inventor tried to using the same strategy of co-expressing a cutinase and an intracellular target protein in a B. subtilis expression system, the effect of extracellular secretion of the target protein is not good.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

ion of Recombinant Plasmid

[0071](1) Plasmid pHYPMLd4P (the plasmid contains pullulanase pμL and chaperone protein prsA genes, and the construction method is recorded in the doctoral dissertation “Modification of Bacillus subtilis Strain, Promoter Optimization and High-Level Expression of Pullulanase”, of Zhang Kang, Jiangnan University, 2018) stored in the laboratory was used as a template to design forward and reverse primers, respectively:

 pHY300PLK-F1:5′-AAGCTTGGTAATAAAAAAACACCTCC-3′;pHY300PLK-R1:5′-TCTTGACACTCCTTATTTGATTTTT-3′;

[0072]An expression vector pHY300PLK-prsA fragment was amplified.

[0073](2) Plasmid xylA / pET24a (+) (the construction method of the plasmid is recorded in Chinese Patent ZL201210581801.2) stored in the laboratory was used as a template to design forward and reverse primers, respectively:

xy1A-F:5′-GGAGTGTCAAGAATGAGCAACTACCAGCCCACAC-3′;xy1A-R:5′-TTTATTACCAAGCTTTTAGCGCACGCCCAGGAGGTAG-3′;

[0074]A xylose isomerase gene fragment was amplified.

[0075](3) The express...

example 2

ion of Cutinase Mutant

[0082]The recombinant plasmid pHY300PLK-xylA-cut obtained in step (4) of Example 1 was used as a template, and according to the gene sequences of cutinase, primers introducing mutations of L175A / T177A, T207A / F209A, I213A / P214A, I178A, L175A, T177A, T207A, F209A, I213A and P214A were designed and synthesized. The cutinase genes were subjected to site-directed mutation and verified by sequencing to obtain recombinant expression vectors containing the cutinase mutant genes: pHY300PLK-xylA-L175A / T177A, pHY300PLK-xylA-T207A / F209A, pHY300PLK-xylA-I213A / P214A, pHY300PLK-xylA-I178A, pHY300PLK-xylA-L175A, pHY300PLK-xylA-T177A, pHY300PLK-xylA-T207A, pHY300PLK-xylA-F209A, pHY300PLK-xylA-I213A, pHY300PLK-xylA-P214A.

[0083]The site-directed mutation primer introducing the L175A / T177A mutation was:

L175A / T177A-F:5′-GATCATCGGGGCCGACGCAGACGCGATCGCGCCGGTCG-3′L175A / T177A-R:5′-CGACCGGCGCGATCGCGTCTGCGTCGGCCCCGATGATC-3′

[0084]The site-directed mutation primer introducing the T207A / F20...

example 3

ion of Recombinant Bacteria Co-Expressing Cutinase Mutant and Xylose Isomerase

[0093](1) Preparation of Competent Cells

[0094]Cryopreserved B. subtilis WS5 was taken by dipping with an inoculating loop, then streaked on an LB solid medium, and cultured overnight at 37° C. for activation. A single colony was picked, inoculated in 10 mL of LB liquid medium, and cultured overnight at 37° C. and 200 rpm for 8 h to obtain a culture solution. 2.5 mL of the culture solution was transferred to 40 mL of LB liquid medium containing 0.5 M sorbitol, and cultured at 37° C. and 200 rpm for 4-5 h to obtain a bacterial solution. The obtained bacterial solution was placed in an ice-water bath for 10 min, and then centrifuged at 4° C. and 5000 rpm for 5 min, and bacterial cells were collected. The bacterial cells were resuspended in 50 mL of a pre-cooled electroporation transformation buffer, and centrifuged at 4° C. and 5000 rpm for 5 min. The supernatant was removed, and the bacterial cells were rins...

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Abstract

Disclosed is a method for promoting extracellular expression of proteins in B. subtilis using cutinase, which belongs to the technical fields of genetic engineering, enzyme engineering and microbial engineering. It teaches co-expressing a cutinase mutant and a target protein in B. subtilis to promote extracellular expression of the target protein which is naturally located inside cells. The target protein includes xylose isomerase, 4,6-α-glucosyltransferase, 4-α-glucosyltransferase, trehalose synthase, branching enzyme and the like. The invention can achieve extracellular expression of intracellularly localized target protein, improve the production efficiency, reduce the production cost and simplify the subsequent extraction process.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims the benefit of priority to Chinese patent application No. 2020112083956, filed Nov. 3, 2020, the content of which is hereby incorporated by reference in its entirety.BACKGROUND OF THE INVENTIONField of the Invention[0002]The invention belongs to the field of genetic engineering, enzyme engineering and microbial engineering. It particularly relates to methods for promoting extracellular expression of proteins in Bacillus subtilis (B. subtilis) using a cutinase.Description of the Related Art[0003]Extracellular expression of exogenous proteins can simplify the downstream purification process, save production costs, and has great advantages in large-scale industrial production. An intracellularly localized protein is expressed inside cells, and the cells need to be disrupted by physical or chemical methods to obtain the target protein. The subsequent extraction process is cumbersome and costly, so it has become the goa...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N9/18C12N9/92C12N9/10C12N15/75
CPCC12N9/18C12Y301/01074C12N9/92C12N15/75C12N9/1051C12Y204/01245C12Y503/01005C12Y504/99016C12N9/1048C12N9/90C12Y204/01161
Inventor WU, JINGSU, LINGQIAHUANG, YAN
Owner JIANGNAN UNIV
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